Smoking is the most common preventable cause of morbidity and mortality in the United States. In efforts to block the initiation of adolescent smoking, a variety of public health measures have been implemented. Despite the success of these measures, a substantial minority of all adolescents still begin the initial 1- 3 year period f experimentation that can lead to regular daily smoking. Effective interventions to block the escalation of this initial smoking exist. However, the implementation and effectiveness of these interventions is hindered by 1) an inability of pediatricians and allied health professionals to detect the presence of the periodic use of cigarettes that characterizes this phase and 2) an inexact understanding of smoking initiation to co-morbid features such as risky sex and alcohol use. This failure deprives a substantial number of adolescents of an opportunity to stop the escalation of smoking and smoking related behaviors during a critical phase of development and education. Recently, we have shown that DNA methylation at AHRR may be a sensitive indicator of early onset adolescent smoking. However, our studies are incomplete in that we have not fully characterized the smoking dose response curve, the effect of other environmental influences, such as second hand smoke, on DNA methylation, and the relationship of DNA methylation to existing indicators of smoking status. In this R01 application, we propose to improve our ability to detect and comprehend the trajectory of smoking behaviors by serially examining a large longitudinal cohort of 450 adolescents at risk for smoking and their families over a two year period. We will determine DNA methylation, serum cotinine and exhaled carbon monoxide levels at multiple time points and exhaustively characterize their environments for potential confounding factors. We will analyze the resulting data with respect to the clinical variables to determine the relationship of DNA methylation to smoking related variables. This application could have high clinical impact, because it may identify a sensitive biomarker of nascent smoking in a population at high risk for complications from smoking and provide a research tool that may allow a wide range of researchers to revisit large existing data sets that focus on youth to investigate health effects of smoking exposure. It is highly feasible because our investigative team has a long history of conducting this type of longitudinal study and we are the discoverers of this biomarker of smoke exposure. It is innovative because unequivocally demonstrated sensitive biomarkers for nascent smoking do not yet exist. The investigative team is led by a well-established physician-scientist and two behavioral interventionists with experience in methylation studies. As a result of this research, we will establish the relationship of AHRR methylation to smoking consumption variables, other biomarker status and environmental exposures (e.g. second hand smoke) that can be used by other for clinical interventions and as a biomarker of smoking exposure for existing DNA collections from epidemiological studies.